Investigating the use of standardized EuroFlow flow cytometry panels for the characterisation and diagnosis of Chronic lymphocytic leukaemia in the Tygerberg Academic Hospital, South Africa.

Musaigwa, Fungai (2017-12)

Thesis (MScMedSc)--Stellenbosch University, 2017

Thesis

ENGLISH ABSTRACT : Background: Flow cytometry (FC) immunophenotyping is crucial in the diagnosis and classification standardisation of haematological malignancies. FC techniques require standardisation to produce reliable and reproducible results which are important in inter-laboratory studies for laboratory methodology improvement. The aim of this study is to introduce standardised multicolour FC in the diagnosis of haematological malignancies using chronic lymphocytic leukaemia (CLL) as the proof of principle. In addition, we aim to document the incidence of CLL from the year 2011 to 2016 in the Tygerberg Academic Hospital (TAH) catchment area of Cape Town, South Africa (SA). Methods: Twenty CLL patients were recruited at TAH. Bio-specimens were prepared and analysed on the Beckman Coulter Navios flow cytometer using Euroflow™ standardised FC protocols and immunophenotypic panels with two tubes for detecting B-cell chronic lymphoproliferative disorders (B-CLPD). Tube 1 included CD20, CD4, CD45, CD8, lg-Kappa, CD56, lg-Lambda, CD5, CD19, TCRyσ, CD3 and CD38. Tube 2 included CD20, CD45, CD23, CD10, CD79b, CD19, CD200 and CD43. Combined, the two tubes identified CLL from other B-CLPD. The CLL immunophenotypic profiles were stored in a database using the compass tool of the Infinicyt™ FC software. In addition, the clinical records of patients diagnosed with CLL at TAH over a 6-year period from the year 2011 to 2016 were retrieved and analysed using descriptive statistics. Results: In comparison with the SA National Health Laboratory Service (NHLS) results at TAH, the Euroflow™ standardised multicolour FC panels and protocols are suitable for immunophenotyping CLL in this SA population. An immunophenotype database for 20 CLL diagnosed at TAH was constructed using the EuroFlow™ standardised multicolour FC panels. For the epidemiology part of the study, a total of 80 CLL patients were studied. There were slightly more females (51.2%) and the mean age at diagnosis was 67 years (37 to 95). Ninety one percent of the patients were aged 50 years and above. Males presented with the disease at a younger age (mean 63 years) than females (mean 70 years). CLL concurrent with HIV was not common (4%) and these patients were younger than 50 years. Twenty-one patients were tested for chromosomal aberrations trisomy 12 and deletion 11q, 24% and 33% were positive respectively. Deletion 13q was assessed in 25 patients and 16% were positive. Twenty patients were tested for deletion 17p and all were negative. Translocations t(8;14), t(11;14) and t (14;18) were negative in 1, 8 and 4 patients respective. Discussion: Accurate and consistent laboratory techniques and strict standardisation in FC enhances the confidence in inter-laboratory studies. Establishment of haematological malignancy immunophenotype databases would allow for faster differential diagnoses of new disease cases which is needed within our setting. Furthermore, these databases permit clear identification of atypical cases. Monitoring haematological malignancy trends is a crucial step in the management of the disease.

AFRIKAANSE OPSOMMING : Agtergrond: Vloeisitometrie (FC) immunofenotipering is van kardinale belang in die diagnose en standaardisering van hematologiese kwaardaardighede/maligniteite. Die FC tegnieke vereis ten einde betroubare en herhaalbale resultate wat belangrik is in inter-laboratorium studies vir die verbetering van laboratorium metodiek. Die doel van hierdie studie was om gestandaardiseerde multi FC in die diagnose van hematologiese maligniteite te gebruik met chroniese limfositiese leukemie (CLL) as bewys van beginsel. Daarbenewens, streef ons daarna om die voorkoms van CLL te dokumenteer in die tydperk van 2011 tot 2016 in die Tygerberg Akademiese Hospitaal (TAH) opvanggebied van Kaapstad, Suid-Afrika (SA). Metodes: Twintig CLL pasiënte was gewerf by TAH. Bio-monsters is voorberei en ontleed op die Beckman Coulter Navios vloeisitometer met behulp van Euroflow™ gestandaardiseerde FC protokolle en immunofenotipe panele met twee buise, vir die opsporing van B-sel chroniese limfoproliferatiewe versteurings (B-CLPD). Buis 1 het CD20, CD4, CD45, CD8, LG-Kappa, CD56, LG-Lambda, CD5, CD19, TCRyσ, CD3 en CD38 bevat. Buis 2 het CD20, CD45, CD23, CD10, CD79b, CD19, CD200 and CD43 bevat. In kombinasie het die twee buise CLL van ander B-CLPD onderskei. Die CLL immunofenotipe profiele is gestoor in 'n databasis met behulp van die kompas instrument (“compas tool”) van die Infinicyt™ FC sagteware. Daarbenewens was die kliniese rekords van pasiënte gediagnoseer met CLL by TAH, oor 'n tydperk van 5 jaar vanaf die jaar 2011-2016, opgespoor en ontleed met behulp van beskrywende statistiek. Resultate: In vergelyking met die SA Nasionale Gesondheids Laboratoriumdienste (National Haleth Laboratory Services - NHLS) resultate by TAH, is die Euroflow™ gestandaardiseerde multi FC paneel en protokolle geskik vir die immunofenotipering van CLL in die SA bevolking. 'n Immunofenotipe databasis vir 20 CLL gevalle, gediagnoseer by TAH, is gebou met behulp van die EuroFlow™ gestandaardiseerde multi FC paneel. Vir die epidemiologiese deel van hierdie studie is 'n totaal van 80 CLL pasiënte bestudeer. Daar was effens meer vroue (51,2%) en die gemiddelde ouderdom by diagnose was 67 jaar (37-95). Een en negentig persent van die pasiënte was 50 jaar en ouer. Mans het die siekte vertoon op 'n vroeër ouderdom (gemiddeld 63 jaar) as vroue (gemiddeld 70 jaar). CLL tesame met MIV was nie algemeen nie (4%) en hierdie pasiënte was jonger as 50 jaar. Een en twintig pasiënte wat getoets was vir chromosoomafwyking, trisomie 12, en verwydering van 11q, was onderskeidelik 24% en 33% positief. Verwydering van 13q is getoets in 25 pasiënte en 16% was positief. Twintig pasiënte was getoets vir verwydering 17p en almal was negatief. Translokasies t (8; 14), t (11; 14) en t (14; 18) was negatief in 1, 8 en 4 pasiënte, onderskeidelik. Bespreking: Akkurate, konsekwente laboratoriumtegnieke en streng standaardisering in FC verhoog die vertroue in inter-laboratorium studies. Die vestiging van immunofenotipe databasisse vir hematologiese maligniteite sal lei tot vinniger differensiële diagnose van nuwe siekte gevalle wat nodig is in ons omgewing. Verder sal hierdie databasisse die duidelike identifisering van atipiese gevalle toelaat. Die monitering van hematologiese maligniteit tendense is 'n belangrike stap in die bestuur van die siektes. (4%) en hierdie pasiënte was jonger as 50 jaar. Een en twintig pasiënte wat getoets was vir chromosoomafwyking, trisomie 12, en verwydering van 11q, was onderskeidelik 24% en 33% positief. Verwydering van 13q is getoets in 25 pasiënte en 16% was positief. Twintig pasiënte was getoets vir verwydering 17p en almal was negatief. Translokasies t (8; 14), t (11; 14) en t (14; 18) was negatief in 1, 8 en 4 pasiënte, onderskeidelik. Bespreking: Akkurate, konsekwente laboratoriumtegnieke en streng standaardisering in FC verhoog die vertroue in inter-laboratorium studies. Die vestiging van immunofenotipe databasisse vir hematologiese maligniteite sal lei tot vinniger differensiële diagnose van nuwe siekte gevalle wat nodig is in ons omgewing. Verder sal hierdie databasisse die duidelike identifisering van atipiese gevalle toelaat. Die monitering van hematologiese maligniteit tendense is 'n belangrike stap in die bestuur van die siektes.

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